Abstract: Background: Debaryomyces hansenii is a highly halotolerant yeast. The objective of this research is to investigate the genes that are involved in Debaryomyces hansenii for tolerance mechanisms against salinity. Methods: In this experiment, the gene DhSSA was cloned from D. hansenii and examined for its expression as induced by salt. The DhSSA gene is orthologous to the Saccharomyces cerevisiae SSA encoded HSP70 which cloned from D. hansenii induced by 2.5 M salt stress and significantly up-regulated during salinity stress. The full-length cDNA of DhSSA has 2,102 bp of nucleotide and contains a 1,926 bp Open Reading Frame (ORF) encoding a deduced protein of 642 amino acid residues (69.9 kDa). Results: Southern blot analysis indicated that DhSSA exists in the genome with one or two copy. Semi-quantitative RT-PCR and Real-time PCR results showed that expression of DhSSA is rapidly induced by salt and its expression increased with time reaching the highest level at 48 min before decline thereafter. Overexpression of DhSSA in Pichia methanolica conferred a higher stress tolerance. Immunocytological labeling reveals that DhSSA is a cytosolic protein. Conclusion: In summary, this study has cloned a salt-induced DhSSA gene from the halophilic yeast D. hansenii by subtraction hybridization. Functional analysis with overexpression transformants in P. methanolica suggest that the gene plays a protective role in the tolerance of D. hansenii to stresses.